Registration Dossier

Administrative data

Description of key information

Oral toxicity: LD50 >32.7 g/kg bw (32700 mg/kg bw).
Dermal Toxicity: LD50 > 16 mL/kg bw (equivalent to 15120 mg/kg bw).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 March 1957 - 27 December 1957
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study pre-dates GLP and the standard guidelines; nevertheless, data is well reported and the experiment is conducted in accordance with generally accepted scientific principles.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
GLP compliance:
no
Test type:
standard acute method
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: young adult.
- Weight at study initiation: 175 - 225 g.
- Fasting period before study: 18 ± 2 hours.
- Housing: Individually caged after dosing.
- Diet: ad libitum.
- Water: ad libitum.
Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Details on oral exposure:
Dosed as a 50 percent solution in cottonseed oil.
Doses:
16.38, 22.62 and 32.72 g/kg bw
No. of animals per sex per dose:
10 animals per sex per dose level.
Control animals:
no
Details on study design:
PROCEDURE
Following the fasting period, the animals were divided into pairs and dosed with the appropriate amount of test material. The highest dose represents the maximum feasible dose for intra-gastric administration.

After dosing, the animals were observed daily for appearance and behaviour. Bodyweight and mortality records were made over a 14 day period.

Rats that died, as well as survivors sacrificed at the end of the experiment, were examined for evidence of gross pathology.
Statistics:
The LD50 was computed according to the method of Miller and Tainter (1944).
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 32.7 other: g/kg bw
Based on:
test mat.
Mortality:
Mortality did not increase in proportion to the dosage.
- 4 deaths (20 %) occurred in the 16.38 g/kg dose group.
- 5 deaths (25 %) occurred in the 22.62 g/kg dose group.
- 3 deaths (15 %) occurred in the 32.72 g/kg dose group.
Clinical signs:
All rats showed evidence of abdominal pain, excessive laxation and urinary incontinence.
Body weight:
The animals gained weight throughout the study.
Gross pathology:
No gross evidence of pathology was seen at necropsy among the survivors sacrificed after 14 days. In the examination of those that died throughout the observation period, the gastric mucosa was observed to be hyperaemic.

Table 1 Summary of Results

Dose (g/kg)

No. of Rats

Average Bodyweight* (g)

Symptoms

(No. of rats exhibiting symptom)

Number of Deaths

Mortality (%)

Days

Days

0

14

A

E

U

P

1

2

3

Over 3

16.38

20

199.0 (198.5)

230.5

20

20

20

2

0

2

0

2

20

22.62

20

203.0 (203.4)

236.4

20

20

20

5

0

3

0

2

25

32.72

20

197.0 (198.8)

234.3

20

20

20

3

0

0

1

2

15

*Parenthetical figures show the average initial weights of the survivors.

†This value was not legible in the original study report. However the value was easily inferred from the percentage mortality figure.

A = abdominal pain

E = excessive laxation

U = urinary incontinence

P = prostration

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the LD50 value was greater than 32.7 g/kg bw. The test material is not classified in accordance with EU criteria.
Executive summary:

An acute oral study was carried out to access the toxic potential of the test material, broadly in accordance with OECD guideline 401.

The test material was administered as a 50 percent solution in cottonseed oil to groups of young adult albino rats at dose levels of 16.38, 22.62 and 32.72 g/kg bw (with ten animals per sex per dose). The animals were observed for 14 days before being subjected to necropsy.

Whilst some mortality was seen, it did not increase in proportion to the dosage. Clinical signs exhibited by all animals were abdominal pain, excessive laxation and urinary incontinence.

No gross evidence of pathology was seen at necropsy among the survivors sacrificed after 14 days. In the examination of those that died throughout the observation period, the gastric mucosa was observed to be hyperaemic.

Under the conditions of this study, the LD50 value was greater than 32.7 g/kg bw. The test material is not classified in accordance with EU criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The findings of the key study have been supported by acute oral toxicity data, on the registered substance, cited in key literature. Additionally acute oral data from several structural analogues have been provided on a read-across basis to support the key study. The overall quality of the dataset is therefore considered to be high.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 March 1957 - 27 December 1957
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study pre-dates GLP and the standard guidelines; nevertheless, data is well reported and the experiment is conducted in accordance with generally accepted scientific principles.
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
The acute dermal toxicity of the test material was investigated by occlusive exposure for 24 hours.
GLP compliance:
no
Test type:
standard acute method
Limit test:
no
Test material information:
Composition 1
Species:
rabbit
Strain:
other: Albino
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Adult.
- Housing: Animals were caged individually.
- Diet: ad libitum.
- Water: ad libitum.
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
- The rabbits were depilated over the entire trunk. Measured volumes of the test material were maintained in contact with the skin by means of a gauze bandage, over which a loose-fitting, impervious plastic sleeve was placed. The sleeve was constricted at both ends in order to prevent leakage of the test sample.
- At the end of the exposure period, the wrappings were removed and the skin wiped with absorbent cotton.
Duration of exposure:
24 hours
Doses:
Doses of 8 and 16 mL (7560 and 15120 mg, respectively) per kg bw.
No. of animals per sex per dose:
1 animal treated at the 8 mL dose level, 10 treated at the 16 mL dose level)
Control animals:
no
Details on study design:
- In a 14 day observation period, records were made of bodyweight, appearance and behaviour.
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 16 mL/kg bw
Based on:
test mat.
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 15 120 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was seen.
Clinical signs:
The appearance and behaviour of the animals was normal throughout. Some transient skin irritation was seen however this was completely reversible within 4 to 7 days.
Body weight:
With the exception of 1 animal, all others maintained or gained weight. This is summarised in Table 1.

Table 1 Net Gain in Bodyweight

Dose

Rabbit No.

Net Gain in Bodyweight (g)

g/kg

mL/kg

7.56

8.0

1

470

 

 

 

 

15.12

 

 

 

 

 

16.0

2

3

4

5

6

7

8

9

10

11

0

100

100

500

300

300

0

-200

100

700

 

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was found to be not systemically toxic to the rabbit at doses of 8 and 16 mL/kg bw (equivalent to 7560 and 15120 mg/kg bw, respectively). The LD50 was considered to be greater than the highest dose tested, 16 mL/kg bw (15120 mg/kg bw). Under the conditions of the study, there is no requirement for the material to be classified in accordance with EU criteria.
Executive summary:

The potential of the test material to cause toxicity via the dermal route was assessed in the albino rabbit.

The depilated trunk of 11 rabbits was exposed to large doses of the test material for a period of 24 hours in an occlusive fashion. A single rabbit was treated with a dose of 8 mL/kg bw; 10 rabbits were treated with a dose of 16 mL/kg bw. Exposure was followed by a 14 day observation period.

No signs of systemic toxicity were observed. Behaviour, appearance and bodyweights (with the exception of 1 animal) were completely normal throughout the study. Some transient skin irritation was observed however this was completely reversed within 4 to 7 days.

 

The test material was found to be not systemically toxic to the rabbit at doses of 8 and 16 mL/kg (equivalent to 7560 and 15120 mg/kg bw, respectively). The LD50 is considered to be greater than the highest dose tested, 16 mL/kg bw (15120 mg/kg bw). Under the conditions of the study, there is no requirement for the material to be classified in accordance with EU criteria.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Quality of whole database:
The findings of the key study have been supported by acute dermal toxicity data provided on two structural analogues. The overall quality of the dataset is therefore considered to be high.

Additional information

Oral Toxicity

The acute oral toxicity has been addressed using a key study performed using the substance to be registered (2,2'-methylenebis(6-nonyl-p-cresol)). The findings of this study have been supported by providing acute oral data cited from key literature and additional studies performed on several structural analogues, provided on a read across basis.

All studies were performed to sound scientific principles with a sufficient level of detail the submitted data. The literature source was cited a peer reviewed handbook. As such all data were assigned a reliability score of 2 in line with principles for assessing data quality as defined in Klimisch (1997).

In the key study (Anon. 1957) the acute oral toxicity of the test material was determined in a study that was performed broadly in accordance with OECD guideline 401. The test material was administered as a 50 percent solution in cottonseed oil to groups of young adult albino rats at dose levels of 16.38, 22.62 and 32.72 g/kg bw (with ten animals per sex per dose). The animals were observed for 14 days before being subjected to necropsy. Whilst some mortality was seen, it did not increase in proportion to the dosage. Clinical signs exhibited by all animals were abdominal pain, excessive laxation and urinary incontinence. No gross evidence of pathology was seen at necropsy among the survivors sacrificed after 14 days. In the examination of those that died throughout the observation period, the gastric mucosa was observed to be hyperaemic. Under the conditions of this study, the LD50 value was greater than 32.7 g/kg bw.

Lewis (2004) has been provided as supporting data, where the LD50 was reported to be 33 g/kg bw in rats, cited directly from the literature.

In the supporting study Birch (1974) the acute oral toxicity of the surrogate substance, 6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol, was determined in male and female Sprague-Dawley rats. The test material was administered at dose levels of 6310 and 7940 mg/kg bw as a 20 % suspension in corn oil. The animals were observed for 14 days and any clinical signs noted. One animal died; reduced appetite and activity were seen for one to three days in survivors. The only gross pathological signs seen were lung and liver hyperaemia and gastrointestinal inflammation in the decedent. The LD50 was determined to be >7940 mg/kg bw.

In the supporting studies Moreno (1986) and Moreno (1983) the acute oral toxicity of the surrogate substance, 2,6-di-tert-butyl-p-cresol, was determined in Wistar rats.

Moreno (1986) was conducted in accordance with the standards set forth by the EPA proposed Guidelines for Test Procedures (1982), Subdivision F. Series 81-1. Ten Wistar rats were dosed orally with the test material at 2000 mg/kg of bodyweight. The rats were observed 1, 2 and 4 hours post dose and twice daily for 14 days for mortality and signs of toxicity. Six animals survived the 2000 g/kg oral dose. Four males died by day 2. Pre-death physical signs were chromorhinorrhea, lethargy, chromodacryorrhea, ptosis, prostration, flaccid muscle tone, diarrhoea, ataxia, piloerection, tremors and soiling of the anogenital area. Physical signs noted in survivors included diarrhoea, piloerection, lethargy, ptosis, chromodacryorrhea, ataxia, chromorhinorrhea, tremors, alopecia, hyperactivity, soiling and brown staining of the anogenital area, soiling of body surfaces, and alopecia of dorsal and ventral surfaces. Necropsy of the deaths revealed abnormalities of the gastrointestinal tract, autolysis of major organs, as well as brown staining of the nose/mouth and anogenital areas. Necropsy of survivors revealed areas of alopecia. The LD50 was determined to be greater than 2000 mg/kg of body weight.

Moreno (1983) was conducted in accordance with the standards set forth by FHSA 16 CFR 1500.3 (c) (2) (i). Initially, ten healthy male Wistar rats were dosed orally with the test material at 5000 mg/kg of bodyweight. Based on the results of the initial dose, ten male rats per group were dosed at 1500, 2000, 2700 and 3700 mg/kg of body weight. A second group of ten rats was dosed with 2000 mg/kg of bodyweight due to odd results seen with the initial dose group. The rats were observed daily for 14 days for mortality and toxicity. Mortality was present in every dose group. Physical signs noted during the study included chromodacryorrhea, piloerection, ptosis, diarrhoea, lethargy, coma, emaciation, dyspnea, yellow nasal discharge, ataxia, prostration, chromorhinorrhea, tachypnea, respiratory abnormalities, soiling and brown staining of body areas, red staining of facial areas and inability to use hind legs. Under the conditions of this study, the LD50 was found to be 2300 mg/kg bw (95 % Confidence limits of 1700 - 3100 mg/kg bw).

In the supporting study Birch (1973) the acute oral toxicity of the surrogate substance, 6,6'-di-tert-butyl-4,4'-thiodi-m-cresol, was determined in male and female Sprague-Dawley rats. The test material was administered at dose levels of 2510, 3160, 3980 and 5010 mg/kg bw as a 25 % solution-suspension in corn oil. The animals were observed for 7 days at which point survivors were terminated and subjected to necropsy alongside any decedents. Mortality was observed at all doses; 1 female in the 2510 mg/kg dose group, 1 female in the 3160 mg/kg dose group, 2 females in the 3980 mg/kg dose group and 2 males and 2 females in the 5010 mg/kg dose group. Clinical signs seen were reduced appetite and activity was seen for 3 to 5 days in survivors, and in the decedents increasing weakness and collapse were observed before death. Gross pathological signs seen were lung and liver hyperaemia along with gastrointestinal inflammation in the decedents; among the animals that survived to the end of the observation period, slight lung congestion was seen in some cases. The LD50 of the test material was 4150 mg/kg bw with 95 % Confidence Limits of 3940 - 4360.

 

Based on the findings of all six studies, it can be concluded that the oral toxicity of the test material is not within the limits of classification.

Inhalation Toxicity

In accordance with line with Regulation (EC) No. 1907/2006 (REACH) Column 2, point 8.5.2, Annex VIII, an acute inhalation study does not need to be performed as the substance has a low vapour pressure (< 0.0015 Pa at 20 °C, determined in the submitted vapour pressure study) and the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur. Furthermore the acute toxicity endpoint has been addressed by assessing the toxicity via the oral and dermal routes, which is more appropriate when considering the properties of this substance.

Dermal Toxicity

Acute dermal toxicity has been addressed using a key study performed using the substance to be registered (2,2'-methylenebis(6-nonyl-p-cresol)). The findings of this study have been supported with acute dermal data on two structural analogues, 6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol and 6,6'-di-tert-butyl-4,4'-thiodi-m-cresol.

In the key study (Anon. 1957) the potential of the test material to cause toxicity via the dermal route was assessed in the albino rabbit. The depilated trunk of 11 rabbits was exposed to large doses of the test material for a period of 24 hours in an occlusive fashion. A single rabbit was treated with a dose of 8 mL/kg bw; 10 rabbits were treated with a dose of 16 mL/kg bw. Exposure was followed by a 14 day observation period. No signs of systemic toxicity were observed. Behaviour, appearance and bodyweights (with the exception of 1 animal) were completely normal throughout the study. Some transient skin irritation was observed however this was completely reversed within 4 to 7 days. The test material was found not to be systemically toxic to the rabbit at doses of 8 and 16 mL/kg (equivalent to 7560 and 15120 mg/kg bw, respectively). The LD50 was therefore considered to be greater than the highest dose tested, 16 mL/kg bw (15120 mg/kg bw).

In the supporting study Birch (1974) the acute dermal toxicity of the surrogate substance, 6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol, was determined using rabbits. The test material was administered at dose levels of 5010 and 7940 mg/kg bw as a 40 % suspension in corn oil to male and female New Zealand albino rabbits. The exposure to the test material lasted 24 hours. The animals were observed for 14 days at which point survivors were terminated and subjected to necropsy alongside any decedents. No mortality was observed; reduced appetite and activity were seen for 2 to 3 days in the survivors. No gross pathological signs were seen, the viscera appeared normal. Under the conditions of this study, the LD50 was determined to be >7940 mg/kg bw.

In the second supporting study Birch (1973) the acute dermal toxicity of the surrogate substance, 6,6'-di-tert-butyl-4,4'-thiodi-m-cresol, was determined in rabbits. The test material was administered at dose levels of 3160, 5010 and 7940 mg/kg bw as a 40 % solution suspension in corn oil to male and female New Zealand albino rabbits. The exposure to the test material lasted 24 hours. The animals were observed for 14 days at which point survivors were terminated and subjected to necropsy alongside any decedents. The male in the high dose group died on day 8. Clinical signs observed in the survivors were reduced appetite and activity for 3 to 7 days; in the decedent, increasing weakness and collapse were seen before death. Gross pathological signs seen were congestion of the lungs and slight liver and kidney discolouration in the survivors. In the decedent, lung hyperaemia, liver discolouration, enlarged gall bladder, discolouration of the spleen and kidneys and gastrointestinal inflammation were seen. Under the conditions of this study, the LD50 was determined to be >5010 mg/kg bw.

All three studies pre-date GLP and the standard guideline; nevertheless, the data are well reported and the experiments were conducted in accordance with generally accepted scientific principles. Accordingly all three studies were assigned a reliability score of 2 in line with the principles of assessing data quality as defined in Klimisch (1997).

 

Based on the findings of all three studies, it can be concluded that the dermal toxicity of the test material is not within the limits of classification.


Justification for selection of acute toxicity – oral endpoint
This is the only avaliable study that addresses the acute oral toxicity of the registered substance. The study pre-dates GLP and the standard guidelines; nevertheless, data is well reported and the experiment is conducted in accordance with generally accepted scientific principles. The study was assigned a reliability score of 2 in accordance with Klimisch (1997).

Justification for selection of acute toxicity – inhalation endpoint
A data waiver has been submitted to address this endpoint.

Justification for selection of acute toxicity – dermal endpoint
This is the only study available that addresses the acute dermal toxicity of the registered substance. The study pre-dates GLP and the standard guidelines; nevertheless, data are well reported and the experiment was conducted in accordance with generally accepted scientific principles. The study was assigned a reliability score of 2 in accordance with Klimisch (1997).

Justification for classification or non-classification

Oral Toxicity

According to the criteria outlined in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, this substance does not meet the criteria for classification for acute oral toxicity.

Dermal Toxicity

According to the criteria outlined in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, this substance does not meet the criteria for classification for acute dermal toxicity.